Blood separation system particularly for concentrating hematopoietic stem cells

ABSTRACT

A system for separating biological fluids into components, comprises a set of containers for the biological fluid to be separated and the separated components, optionally an additional container for additive solution, and a hollow centrifugal processing chamber having an axial inlet/outlet for the biological fluid. The processing chamber contains a piston movable to intake a selected quantity of biological fluid and express processed biological fluid components via the outlet. Optical means monitor the position of piston to control the amount of intaken fluid and the expression of components. A distribution valve arrangement selectively communicates the processing chamber and the containers or places them out of communication. The system is arranged to operate in a seperation mode and in a non-separation transfer mode, especially for adding preservative solution to separated blood stem cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the national phase application of InternationalApplication No. PCT/IB99/02053 filed Dec. 24, 1999, entitled “BloodSeparation System Particularly for Concentrating Hematopoietic StemCells.” Priority is claimed to the PCT application filing date under 35U.S.C. § 365.

FIELD OF THE INVENTION

This invention relates to the automated processing and separation ofbiological cells as found in whole blood, and relates more specificallyto a functionally closed system allowing to extract certain cellpopulations like hematopoietic stem cells, for immediate use or theirmixing with an additive solution or a storage solution for laterseparate storage operations and to the methods for carrying out such anextraction.

BACKGROUND OF THE INVENTION

Blood separation systems and methods have emerged over the past 20 yearsin response to the growing need for efficient blood component therapies.Among them are the transplantation of hematopoietic progenitor stemcells, which in many cases is the only remaining cure to oncologicaldisorders. Patients in need of a stem cell transplant have mainly threeoptions:

1) Adult bone marrow stem cells;

2) Peripheral blood stem cells found in the circulatory system;

3) Stem cells found in umbilical cord and placental blood retrieved atbirth of a new born infant.

For most stem cell transplants, the main limitation has been the risk ofgraft-versus-host-disease (GVHD), requiring an excellent HLA-matchtissue (HLA=Human Leucyte Associated).

Umbilical cord blood is a rich source of the primitive hematopoieticstem and progenitor cells, with extensive proliferation capacity andcapacity to self-renew. This field has advanced rapidly from clinicalimplants utilizing only HLA-matched grafts to unrelated donor celltransplants which open a much larger indication for stem celltransplantation. This increase in clinical experience with cord blood isdue mainly to the establishment of banks for storage of hematopoieticstem cells from unrelated umbilical blood cord.

Blood volumes recovered from umbilical cord are usually very low (40 to150 ml) and there is some concern that any attempt at productmanipulation and concentration might result in stem cell loss, whichmight impair engraftments. Therefore umbilical cord blood is sometimesstored as is, with preservative solution added. A much preferred waywould be to eliminate most unwanted cells like red cells and whitecells, resulting in a considerable volume reduction. Less preservativesolution would be required, smaller bags, smaller storage spaces wouldbe used and considerable energy savings achieved, all of thistranslating in substantial cost savings. The quality of the stem cellproduct when retransfused would be improved as well, as lysed cellsresulting from storage would be drastically reduced.

No device or automated system exists for processing and concentrating online umbilical cord stem cells. There is nevertheless a considerableinterest for concentrating umbilical cord blood stem cells without lossor altering their functionality.

EP-B-0 912 250 (C. FELL), the contents whereof are herein incorporatedby way of reference, describes a system for the processing andseparation of biological fluids into components, comprising a set ofcontainers for receiving the biological fluid to be separated and theseparated components, and optionally one or more additional containersfor additive solutions. A hollow centrifuge processing chamber isrotatable about an axis of rotation by engagement of the processingchamber with a rotary drive unit. The processing chamber has an axialinlet/outlet for biological fluid to be processed and for processedcomponents of the biological fluid. This inlet/outlet leads into aseparation space of variable volume wherein the entire centrifugalprocessing of biological fluid takes place. The processing chambercomprises a generally cylindrical wall extending from an end wall of theprocessing chamber, this generally cylindrical wall defining therein thehollow processing chamber which occupies a hollow open cylindrical spacecoaxial with the axis of rotation, the axial inlet/outlet being providedin said end wall coaxial with the generally cylindrical wall to openinto the hollow processing chamber. The processing chamber containswithin the generally cylindrical wall an axially movable member such asa piston. The separation space of variable volume is defined in an upperpart of the processing chamber by the generally cylindrical wall and bythe axially movable member contained in the generally cylindrical wallof the processing chamber, wherein axial movement of the movable membervaries the volume of the separation space, the movable member beingaxially movable within the processing chamber to intake a selectedquantity of biological fluid to be processed into the separation spacevia the inlet before or during centrifugal processing and to expressprocessed biological fluid components from the separation space via theoutlet during or after centrifugal processing. Means are provided formonitoring the position of the movable member to thereby control theamount of intaken biological fluid and the expression of separatedcomponents. The system further comprises a distribution valvearrangement for establishing selective communication between theprocessing chamber and selected containers or for placing the processingchamber and containers out of communication.

The system according to EP-B-0 912 250 is designed to operate for theseparation of biological fluids, and has proven to be very polyvalentfor many separation applications, especially for on-line separation ofcomponents from a donor or a patient.

DISCLOSURE OF THE INVENTION

According to the invention, such system is arranged to operate in aseparation mode and in a non-separation transfer mode, which providesgreater possibilities for use of the system including new applicationswhich were heretofore not contemplated, such as separation ofhematopoietic stem cells and in general laboratory processing. Accordingto the invention, the system is arranged to operate such that:

in the separation mode fluids can be intaken into the processing chamberwhile the chamber is rotating or stationary, fluid intaken into thechamber is centrifuged and separated into components, and the separatedcomponents expressed while the chamber is rotating or, optionally, forthe last separated component, while the chamber is stationary; and

in the transfer mode the processing chamber intakes fluid and expressesfluid with the chamber stationary, The valve actuation arrangement isactuable to transfer amounts of fluid from one container to another viathe processing chamber, by moving he member, without centrifugation orseparation of the fluid into components, and the means for monitoringthe position of the movable member controls the amounts of non-separatedfluids transferred.

Further features of the invention are set out in the claims. Thisinvention thus proposes a functionally closed processing kit associatedwith a portable apparatus, whose function is to monitor and automate theprocedure. The kit, usually disposable for avoiding the likelihood ofdisease transmission, is based on a centrifugal processing chamber whosevolume can be varied during operation, allowing to adjust to the exactquantity of blood to process. Such variable volume chamber is describedin the aforementioned EP-B-0 912 250 (C. FELL). The chamber is connectedto a set of bags and tubing lines for the collection of the separatedcomponents. The blood bag containing the blood to process is generallyconnected to the disposable set through the use of a sterile connectingdevice, or an aseptic connection under laminar flow. It is howeverpossible to have this bag prefilled with anticoagulant and preconnectedto the disposable kit.

A bag containing an additive solution can be connected to the disposablekit via a bacterial filter. The other bags are provided for thecollection of the separated components. The stem cell collection bagmaterial is optimally chosen for the storage conditions.

The tubing line selection for conveying the separated products into theproper bags is accomplished by a set of rotational valves calledstopcocks that can be arranged in a manifold array, or by a singlemultiport rotational valve, forming part of the set. Such an arrangementallows to eliminate any cross-contamination between adjacent lines whenusing standard pinch valves.

The above-mentioned disposable kit cooperates with an instrumentationfor monitoring and automating the process, for instance as described inEP-B-0 912 250 (C. FELL). The centrifuge drives a rotating disk whichreceives the centrifugal processing chamber and locks it in place. Itsclosing cover will grip and hold she housing of the rotary seal of theprocessing chamber.

An optical sensor made of an array of LED and corresponding receivingsensors placed at 180° is implemented vertically on the side ofcentrifuge, for monitoring the piston position. Volumes intaken into orextracted from the chamber can therefore be exactly measured. Thetopdeck receives an optical line sensor module which monitors the colorin the effluent tubing, feeding-back this information to the controlprogram. An array of shafts equipped with fittings for driving a set ofmultiple stopcock valves protrude from the top deck. They are coupled toa set of motors enabling the tubing line selection. Encoders areattached to the motors for monitoring the stopcock valves position. Thefront panel incorporates a window allowing the user to see displacementof the piston in the chamber.

The procedure for extracting stem cells out of an umbilical cord is asfollows. Initially blood is recovered from the umbilical cord at birthand collected aseptically into a plastic bag, with anticoagulant addedlike Citrate-Phosphate-Dextrose CPD-1 to avoid clotting. After initialsampling is taken to assess its richness in stem cells, the bag issterile or aseptically connected to the processing kit and the whole setis loaded onto the separation system, which initially operates in theseparation mode or the transfer mode at choice. In the separation modethe centrifuge starts driving the separation chamber at around 4000 rpm,and blood is introduced by moving down the chamber piston pneumatically.Two cases can then occur. If the volume of blood to process is smallerthan the processing chamber volume (as detected by the empty state ofthe effluent tubing), the piston is maintained at an intermediateposition pneumatically, monitored by the piston position sensor. If thevolume of blood completely fills the separation chamber, detected by thepiston reaching the bottom of the chamber, the pneumatic compressorstops. In both cases, centrifugation speed is increased to around 6000rpm to shorten the sedimentation time to 5-8 min. After this period,centrifugation slowly decreases to around 4000 rpm. Stopcocks arerotated to allow the collection of the separated products, and thepneumatic pressure gradually increases to move the piston upwards. Thespeed of the piston remains low, actively monitored by she pistonposition sensor, in order to maintain the sedimentation profile of thecells within the chamber. The first milliliters from the inlet line arepurged in the stem cell bags. Plasma is extracted then collected into afirst bag. It is followed by platelets, packed in the intermediatelayers, or buffy-coat. Apparition of the first platelets is detected bythe optical line sensor monitoring the effluent line tubing. At thatmoment, the product, very rich in stem cells, is directed into a secondcollection bag by rotating a stopcock valve. A volume counter isstarted, which depends among other factors on the total blood volumeprocessed. When this counting volume has been reached, centrifugation isstopped. The proper stopcock valve is rotated and the last product isextracted, essentially a volume of packed red cells with residualgranulocytes, into a third collection bag. Another cycle can then resumeif the umbilical cord blood has not been totally processed. Otherwise,the separation and stem cells collection process is completed at thisstage.

However, it is possible to reprocess the content of the bag containingthe stem cells, in view of further purifying the product. In this case,the proper stopcock valve is rotated to intake the content of the stemcell concentrate bag. The procedure to collect stem cell rich layer isidentical then to the one described above.

Another alternative to isolate the stem cell rich fraction from thebuffy-coat is by using density gradient products such as those availableunder the names Ficoll and Percoll. In this alternative, a densitygradient product is first introduced into the processing chamber,followed by introduction of whole blood, and a component of thebiological fluid is separated into a giver container and its collectionis completed when the density gradient appears. Possibly the densitygradient product may be introduced during processing.

Using Ficoll would for example consist of first introducing the densitygradient into the processing chamber, followed then by whole blood.After complete introduction of blood into the chamber, a sedimentationperiod of a few minutes is started. Stem cells and platelets form aninterface in front of the gradient, whereas erythrocytes andgranulocytes have passed through the Ficoll and are held against thewalls of the separation chamber. The piston is then lifted gently as inthe standard procedure, the stem cell fraction being collected at theapparition of the first platelets. The effluent line clears up againwhen Ficoll exits the chamber, which is the appropriate moment to stopthe collection.

When the stem cells are collected by one of the methods described aboveadequate preservative solution can be introduced into the processingchamber by rotating the proper stopcock, the system operating in thetransfer mode. It is then retransferred into the stem cell bag, itsvolume accurately controlled by the piston position sensor.

The bag containing the stem cell rich product can be disconnected atthis stage from the rest of the set. Its volume ranges between 20-40 ml,depending of the initial volume processed. The by-products of theseparation, plasma and packed red cells, can then be used for serologyand HLA typing, avoiding any product loss due to sampling in the stemcell bag.

This separation system and method offer significant advantages overmanual processing techniques. The disposable kit is a functionallyclosed system, avoiding any risk of contaminating the product duringmanipulation. The protocol is fully automated, through a microprocessorbased control system, with ability to vary the main parameters, likecentrifugation speed, centrifugation time, speed of introduction andextraction, volume to collect, etc. The volume reduction for the stemcell product represents a gain of 50% at least compared with the currentstate of the art. The instrumentation is very compact and portable,ideal for the decentralized processing of such procedures.

A further aspect of the invention is the use of the above-describedsystem for processing variable volumes of biological fluid from 10 ml upto the maximum volume of the separation chamber, and for adding anadditive solution to the separated components, in particular forseparation of stem cells from blood and mixing the separated stem cellswith a preservative solution; for separation of hematopoietic stem cellsfrom umbilical cord blood; for separation of hematopoietic stem cellsfrom an apheresis collection; and for separation of hematopoietic stemcells from a bone marrow aspirate.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention Will be further described by way of example with referenceto the accompanying drawings in which:

FIG. 1 is a schematic side elevation and cross-sectional view of aprocessing chamber and its rotary seal, showing the varioussedimentation layers of blood components.

FIG. 2 is a schematic side elevation and cross-sectional view of theprocessing chamber and its rotary seal, associated with an opticalsensor for monitoring the piston sensor, and control circuitry.

FIG. 3 illustrates in a schematic form the disposable set carrying amanifold stopcock system for the processing and separation of umbilicalcord blood.

FIG. 4 illustrates in a schematic form an array of stopcocks arranged ina manifold.

FIG. 5 is a schematic side elevation and cross-sectional view of themotor driver and control elements for rotating each individual stopcock.

FIG. 6 is a schematic side elevation and cross-sectional view of amultiport rotational valve.

FIG. 7 schematically illustrates the multiport rotational valve of FIG.6 with an inlet/outlet port located at the center and a range ofassociated ports located at the periphery.

FIG. 8 is perspective view of a cabinet containing instrumentation anddevices for controlling the processing.

FIG. 9.1 to FIG. 9.6 are functional diagrams showing the various stepsof umbilical cord blood separation using a disposable set including aprocessing chamber and a set of stopcocks according to the invention.

DETAILED DESCRIPTION OF THE SYSTEM

The processing chamber 20 is in accordance with that described in EP-B-0912 250 (C. FELL). FIG. 1 is a general view of the processing chamber20. A rotary seal 12 is located on its upper extremity 10. The rotaryseal 12 is composed of an upper body 1 and lower body 2. In between islocated a friction disk 3, made of a generally low friction materiallike polished stainless steel or ceramic. A central tubing 7 made ofbiocompatible material like polycarbonate is attached to the upper body1. An O-ring 8 ensures airtightness between the upper body 2 andfriction disk 3. The rotary seal 12 is mounted on a central bush 11fitted on the upper extremity 10 of processing chamber 20. However,central bush 11 can be an integral part of chamber 20. The gap betweenwalls of central tubing 7 and central bush 11 is small, say 0.5 mm, toprovide a high rotational impedance for stopping any liquid to reach theupper extremity of bush 11. A ball bearing 9 is mounted on bush 11 toensures the proper alignment of the processing chamber 20 when insertedinto centrifuge assembly. Two rubber seals 5, 6 are located on eitherside of the friction disk 3, seal 5 being on the upper side and seal 6being on the lower side. The seals 5 and 6 are of the V-seal type andensure airtightness both in positive and negative pressure, up to atleast +−0.5 bar.

The piston 21 is made of a transparent material like polycarbonate andis equipped with two O-rings 24 and 25. These O-rings are made of lowfriction material like silicon. The processing chamber 20 is closed onits bottom side by a cap 22 carrying a bacterial filter 23. Air can passthrough central opening 26 and filter 23 in cap 22. The position of thepiston 21 can be accurately monitored by an optical sensor assembly 60and 61 (FIG. 2). Assembly 61 is made of a vertical array of LED,preferably with light emitting in the infrared spectrum to reducedisturbance from ambient light. Only the LED facing piston 21 are turnedon, in order to avoid interference from the other LED. The beam of lightcrosses the transparent piston 21, between the two O-rings 24 and 25. ACCD (“Charge Coupling Device”) linear array 61 is placed at 180° on theother side, the exposed pixels of array 61 generating a signal 62 in theform of a peak.

Signal 62 is fed to a low-pass filter 69 and the filtered signal fed toa comparator 67 which also receives from potentiometer 68 a thresholdvalue for discriminating the filtered signal from ambient noise. Theoutput of comparator 67 is connected to the enable gate of counter 65.Clock signal 66 is used to intake the response from each individualpixel of the CCD linear array 61, and feed this to the input of counter65. The output of counter 65 is connected to a CPU 64 which calculatesthe position of piston 21 and, when required, shifts the turned-on LEDs60 via a multiplexer/LED driver 63. Similarly, when necessary, the CPU64 will vary the signal of compressor driver 70 that supplies compressor71 in order to increase or decrease pressure applied below the piston 21to control its position.

This is only one example of position sensing for the piston 21. Thelight source 60 could be a filament bulb, or a unique linear source oflight. The CCD linear array 61 could be replaced by an array ofphotosensing devices. The receiving sensing device (61) could be placedalso beside the emitting light device 62, the system working inreflection light from the piston 61 rather in transmittance lightthrough the piston 21.

The disposable Bet (FIG. 3) is composed of bags 40-44, tubing linesconnected to stopcocks 45-48, and the processing chamber 20. Bag 40contains the umbilical cord blood to process. Bag 41 containspreservative solution, generally based on a DMSO (Dimethyl Sulfoxide)solution. It is connected to the disposable set through a bacterial 0.2micron filter 54. Bag 42 is the collection bag for the stem-cell richproduct. Its plastic composition is made of a material suiting long termstorage. Bag 43 is the collection bag for the plasma and bag 44 is theone for the red cells.

FIG. 3 also shows means for rotating the chamber 20 by contacting thechamber's bottom 22 with a rotary disc 55 without any support at thechamber periphery.

An array of stopcocks 45-48 (FIG. 5) organized in a manifold 58 allowsthe connection between the different tubing lines. These rotationalstopcocks provide an excellent cut-off between adjacent lines and ensurethat no leak occurs between a closed and an open line, as is the casewith tubing pinch valves. Such manifold stopcocks exist in various formsand are commercially available. The stopcocks 45-48 are driven by a setof motors 100-103. (FIG. 5). The upper shafts 84-87 of these motorsengage into the bottom portion of the respective stopcocks 45-48, usingpassing holes through the cabinet topdeck 88. As a safety measure toallow for possible manual actuation, the shafts can engage into thestopcock in one position only, a matching indent being provided for thispurpose between the shafts and the stopcock. The motors can be steppermotors or DC motors with reductors. They are equipped with positionencoders 104-107, whose signals are fed back to the microprocessorcontrol unit, ensuring that the stopcocks are correctly positioned.

An alternative to using a manifold stopcock is a rotational multiportvalve, as shown in FIGS. 6 and 7. A central rotor 127 is inserted into astator 126. The rotor 127 can be frictionally rotated and can be engagedon the shaft of a motor. The central port 120, connected to theprocessing chamber 20, can be connected to the peripheral ports 121-125by controlled rotation with angular steps or 720. As a safety measure toallow for possible manual actuation, matching indents or other means canbe provided in the rotor 127 and stator 126 for snap-holding the centralport 120 in its selected angular positions aligned with the peripheralports. A single motor is necessary to drive the rotor through theengagement recess 132. Two O-rings 130-131 ensure watertightness withthe exterior (FIG. 6).

The cabinet holding the instrumentation is shown in FIG. 8. It containsthe cover 94 for holding the rotary seal 12 of processing chamber 20.The cover 94 is made of two semi-circular disks that can rotate on hinge89. An optical line sensor 83 allows the sensing of colors in theeffluent tubing 51. It holds two LED-Photosensor channels, of differentwavelength like red and green, and is capable of detecting the firstcells coming out of the chamber 20. It can equally detect the emptystate of the effluent line tubing when liquid is introduced in thechamber. The pressure port measurement 93 receives the bacterial filter49 located on the disposable set. This allows the monitoring of thepressure in the processing chamber 20. Upper shafts 84-87 of thestopcock driver motors 100-103 (FIG. 5) are located behind the linesensor 83. An inclined module 90 receives the display 82 for userinformation and a keyboard 81 for controlling the instrumentation. Awindow 91 is located on the front panel 92 giving visibility to thechamber piston movement.

Application for Umbilical Blood Separation

FIGS. 9.1 to 9.6 illustrate an application for umbilical cord bloodseparation. Bag 40 contains the umbilical cord blood rich in stem cells,recovered from the umbilical cord at birth of a child.

This bag 40 contains anticoagulant like CPD to avoid blood clots. Tubingline 53 is sterily connected to line 52 using a sterile connectingdevice or aseptically connected under laminar flow. However it is alsopossible to have the bag 40 preconnected to the whole set. Theseparation steps are:

Step 1 (FIG. 9.1): Stopcocks 45 and 46 are rotated to connect bag 40 tothe processing chamber 20. Centrifugation starts and initially isstabilized at a speed of 4000 rpm. The pneumatic system of theinstrumentation establishes a vacuum to move the piston 21 downwards.Its speed is monitored by the optical sensor assembly 61 and 62, and thevacuum level is adjusted accordingly. If the volume of the bag 40 issmaller than the processing volume of chamber 20, effluent tubing line51 will empty, which is detected by the optical line sensor 83. Thepiston 21 is maintained motionless by establishing a counter pressurethrough the pneumatic system, and the volume intakes into chamber 20 isrecorded. The centrifuge speed is gradually increased to reach 6000 rpm,with pressure increased accordingly to keep piston 21 at the sameposition. In the case where the volume of bag 40 is larger than theprocessing volume of chamber 20, piston 21 will reach the bottom of thechamber, and the pneumatic system is turned off. In both cases, after asedimentation time of about 5-8 min, centrifuge speed is slowlydecreased, while maintaining a constant counter pressure below piston21. Stopcocks 46, 47, 48 are rotated to establish a path between theprocessing chamber 20 and the plasma bag 43.

Step 2 (FIG. 9.2): When the centrifuge drops to a speed generally around4000 rpm, the piston 21 starts to move upwards, with a preset speedallowing extraction rates around 100 ml/min. This value can be modifiedthrough the program parameters. Plasma starts to be collected in bag 43.When the volume of plasma extracted reaches approximately 40% of thevolume intaken, the extraction rate will be reduced by half. The firstplatelets contained in the buffy-coat layer start to be extracted, whichis detected by light absorbance in the optical line sensor 83. At acertain level of absorbance, which can be parametered by the controlprogram, stopcock 47 is rotated to establish the path between theprocessing chamber 20 and bag 42.

Step 3 (FIG. 9.3): A volume counter is triggered, and collection of aproduct very rich in stem cells starts. Extraction speed is always underthe control of the piston optical sensor assembly 61 and 62. The volumecounter value can be altered by the user in the program menu. It ischosen in order to encompass all the stem cell population, which hassimilar characteristics in density and size to the lymphocytepopulation. Such value corresponds generally to 20-30% of the intakenvolume into the chamber. When this value is reached, stopcocks 47 and 48are rotated to establish a path with bag 44.

Step 4 (FIG. 9.4): Centrifugation is generally stopped at this stage,and pressure diminished to allow a smooth extraction of the remainingred cells into bag 44. This phase is completed when piston 21 reachesthe top of the processing chamber 20, detected by the piston opticalsensor assembly 61 and 62. At this stage, if the bag 40 is not empty,the process will be resumed at Step 1, otherwise it will proceed withStep 5, which is the transfer mode. An optional phase is to furtherseparate the stem cell product by returning the content of bag 42 intothe processing chamber 20, centrifuging the product once again andexpressing its separated components to bags 43, 42 and 44 as before.

Step 5 (FIG. 9.5): Stopcocks 45 and 46 are rotated in order to establisha path between the preservative solution bag 41 and the processingchamber 20. The preservative solution is generally a composition basedon 10 or 20 vol % DMSO chemical solution, which can contain also aphosphate buffer. The centrifuge being idle, piston 21 is moveddownwards by establishing a vacuum with the pneumatic system. The volumeintaken is a proportion of the volume counter described in Step 4. Whenthis proportion is reached the vacuum stops and stopcocks 46 and 47 arerotated in order to establish a path between the processing chamber 20and the stem cell bag 42.

Step 6 (FIG. 9.6): The pneumatic system is turned down, and piston 21moved upwards. The preservative solution is added to the content of thestem cell bag 42. This transfer phase is completed when the pistonreaches the top of the processing chamber 20, detected by the pistonoptical sensor assembly 61 and 62. An optional additional phase can beadded if the stem cell product needs to be diluted further with plasma.In this case Steps 5 and 6 will be repeated, with the difference thatthe transfer will be established between the plasma bag 43, theprocessing chamber 20 and the stem cell bag 42.

When all the steps described above are completed, all the stopcocks canbe rotated at a 45° angle in order to close all the communicating ports.The bags 42-44 can be disconnected from the rest of the set, which canbe discarded at this stage. The stem cell bag 42 is then ready forconveying to a separate storage unit, the by-products plasma in bag 43and red cells 44 being used for HLA typing and quality controlassessments.

It will be appreciated that including a transfer mode, i.e. steps 5 and6, opens up new applications for the system that were not available whenthe apparatus operated solely in the separation mode, in particular forapplications requiring adding an additive solution to the separatedcomponents.

It is to be understood that this invention may be embodied in severaldifferent forms without departing from its spirit of essentialcharacteristics. The scope of the invention is defined in the appendedclaims, rather than in the specific description preceding them. Allembodiments that fall within the meaning and range of equivalency of theclaims are therefore intended to be embraced by the claims.

Moreover, the novel optical control device (60-71) herein described, aswell as the arrangement of stopcocks (45-48) and tubing, the multiportvalve (FIGS. 6 and 7), the special rotatable seal (1-7) for positive andnegative pressure operation, and the special axial bearings mounting thechamber 20, permitting drive without a holding chuck, can alladvantageously be used in different systems.

What is claimed is:
 1. A system for the processing and separation ofbiological fluids into components, comprising a set of containers forreceiving the biological fluid to be separated and the separatedcomponents, and optionally one or more additional containers foradditive solutions, and a hollow centrifugal processing chamberrotatable about an axis of rotation and having an axial inlet/outlet forthe biological fluid to be processed and for the processed components ofthe fluid, the processing chamber containing an axially movable memberwhich defines a separation space of variable size for receivingbiological fluid, the member being axially movable to intake a selectedquantity of biological fluid to be processed into the separation spacevia said inlet and to express processed biological fluid components fromthe separation space via said outlet, and means for monitoring theposition of the axially movable member to thereby control the amount ofintaken biological fluid and the expression of separated components, thesystem further comprising a distribution valve arrangement forestablishing selective communication between the processing chamber andselected containers or for placing the processing chamber and containersout of communication, the system comprising means for controllingoperation of the system in two operational modes, a separation mode anda non-separation transfer mode, wherein: in the separation mode fluidscan be intaken into the processing chamber while the chamber is rotatingor stationary, fluid intaken into the chamber is centrifuged andseparated into components, and the separated components expressed whilethe chamber is rotating or, optionally, for the last separatedcomponent, while the chamber is stationary; and in the transfer mode theprocessing chamber intakes fluid and expresses fluid with the chamberstationary, the valve actuation arrangement being actuable to transferamounts of fluid from one container to another via the processingchamber, by axially moving the member, without centrifugation orseparation of the fluid into components, and said means for monitoringthe position of the axially movable member controls the amounts ofnon-separated fluids transferred.
 2. The system of claim 1, wherein thedistribution valve arrangement comprises a set of rotational stopcockvalves arranged in a manifold array, or a multiport rotational valve. 3.The system of claim 1, wherein the distribution valve arrangementcomprises a plurality of stopcock valves connected to tubing linesinterconnecting the set of containers, the optional additionalcontainers, the processing chamber and further stopcock valves, eachstopcock valve comprising a rotatable stopcock valve member having ashaft associated with drive means, said shaft being rotatable toselectively connect or disconnect the stopcock valve's tubing lines. 4.The system of claim 3, comprising means for allowing insertion of eachstopcock valve only in a defined angular alignment of the rotatablestopcock valve member.
 5. The system of claim 1, wherein thedistribution valve arrangement comprises a multiport valve comprising acentral rotor rotatably mounted in an annular stator, the rotor having acentral port connected to the processing chamber and leading to therotor outer periphery, and the stator having a plurality of ports atselected angular locations each connected to a container and eachleading into the inner periphery of the annular stator, the central portof the rotor being connectable to selected ports of the stator, ordisconnected, by rotation of the rotor.
 6. The system of claim 1,wherein the movable member is a piston fluid-tightly movably mounted ina generally-cylindrical centrifugal processing chamber.
 7. The system ofclaim 6, further comprising optical means for monitoring the position ofthe piston, comprising an alignment of light emitting elements generallyparallel to the piston axis, and an alignment of light receivingelements generally parallel to the piston axis, the receiving elementsbeing arranged to receive light from the emitting elements transmittedthrough or past the piston or reflected by the piston, and to deliver asignal representative of the piston's position.
 8. The system of claim7, wherein the receiving elements are arranged to deliver said signal tomeans for moving the piston and means for controlling the piston'sposition.
 9. The system of claim 1, comprising an optical sensormonitoring fluid in the tubing line connected to the axial inlet/outlet,for stopping the intake of biological fluid when the tubing line isempty during the intake mode and/or for providing a signal for switchingthe distribution valve arrangement in the extraction mode.
 10. Thesystem of claim 1, wherein the axial inlet/outlet comprises a rotatableseal mountable in a stationary housing, said seal being operable forpositive and negative pressure conditions in the rotatable chamber. 11.The system of claim 1, wherein the processing chamber is mounted forrotation about its axis by means of bearings at opposite ends of thechamber, one end of the chamber being associated with means for rotatingthe chamber by contacting the chamber's bottom with a rotary discwithout any support at the chamber periphery.
 12. The system of claim 1,wherein the means for controlling operation of the system in said twooperational modes comprises a microprocessor based control systemcontrolling an automated protocol.
 13. A method of processing andseparating biological fluids in a system according to claim 1, themethod comprising: separating a biological fluid with the systemoperating in the separation mode, by intaking fluid into the processingchamber while the chamber is rotating or stationary, centrifuging fluidintaken into the chamber to separate the fluid into components, andexpressing the separated components while the chamber is rotating orpossibly, for the last component, while the chamber is stationary; andtransferring fluid between containers with the system operating in thetransfer mode, by intaking fluid into the processing chamber with thechamber stationary, actuating the valve distribution arrangement totransfer an amount of fluid from one container to another via theprocessing chamber, by moving the member, without centrifugation orseparation of the fluid into components, and monitoring the position ofthe movable member to control the amount of non-separated fluidtransferred.
 14. The method of claim 13, wherein a component of thebiological fluid is separated into a given container, the amount of saidcomponent separated into the given container being controlled bymonitoring the position of said member, and an additive solution istransferred from an additional container to said given container via theprocessing chamber in said transfer mode, the amount of additivesolution transferred being calculated as a function of the amount ofsaid separated component in the given container.
 15. The method of claim13, wherein a density gradient product and blood are introduced into theprocessing chamber, and a component of the biological fluid is separatedinto a given container and its collection is completed when the densitygradient appears.
 16. The method of claim 13, wherein operation of thesystem in said two operational modes is controlled according to anautomated protocol by a microprocessor based control system.
 17. Adisposable set for collecting and separating selected quantities ofbiological fluids comprising the centrifugal processing chamber of asystem according to claim 1, wherein the inlet/outlet of the centrifugalprocessing chamber is connected to a container of biological fluid, anadditional container containing an additive solution, a plurality ofcontainers for receiving the separated components of the biologicalfluid, interconnected by a distribution valve arrangement comprising aset of rotational stopcock valves arranged in a manifold array, or amultiport rotational valve.
 18. The disposable set of claim 17, whereinthe distribution valve arrangement comprises a plurality of stopcockvalves connected to tubing lines interconnecting the set of containers,the optional additional containers, the processing chamber and furtherstopcock valves, each stopcock valve comprising a rotatable stopcockvalve member having a shaft associated with drive means, said shaftbeing rotable to selectively connect or disconnect the stopcock valve'stubing lines.
 19. The disposable set of claim 17, wherein thedistribution valve arrangement comprises a multiport valve comprising acentral rotor rotatably mounted in an annular stator, the rotor having acentral port connected to the processing chamber and leading to therotor outer periphery, and the stator having a plurality of ports atselected angular locations each connected to a container and eachleading into the inner periphery of the annular stator, the central portof the rotor being connectable to selected ports of the stator, ordisconnected, by rotation of the rotor.
 20. The method of claim 14,wherein variable volumes of biological fluid from 10 ml up to themaximum volume of the separation chamber are processed and an additivesolution is added to the separated components.
 21. The method of claim13, wherein the fluid is blood and the separated component is stem cellsand a preservative solution is mixed with the stem cells.
 22. The methodaccording to claim 21, wherein the stem cells are hematopoietic stemcells from umbilical cord blood, from an apheresis collection, or from abone marrow aspirate.